High resistance of bacteria to antibiotics and detrimental changes to beneficial microbiome caused by frequent use of antibiotics require development of new approaches to antibacterial therapy. One promising target for such a new approach is bacterial stress responses, whose activation by human innate immunity proteins, Peptidoglycan Recognition Proteins (PGRPs), kills bacteria. The responses evoked by PGRPs in bacteria include oxidative, thiol, and metal stress, which synergistically kill bacteria by eventual shutdown of all biosynthetic reactions. The long-term goal of this project is to attain successful targeting of bacterial stress responses in antibacterial therapy and prevention by enhancing the functions of endogenous host PGRPs or by developing PGRP analogs or mimetics. However, it is not known how PGRPs induce these stress responses, what the sequence of these responses is, which responses kill bacteria and how, and which responses defend bacteria against killing. This knowledge gap is an obstacle to successful targeting of bacterial stress responses as a new approach to antibacterial therapy. The objective of this project is to fill this knowledge gap by testing the hypothesis that the following sequence of events in bacteria is responsible for PGRP-induced killing (which is based on currently available data): PGRP binding to cell wall or outer membrane ? activation of regulators of multiple stress responses ? increase in Crp- and Arc-controlled TCA cycle ? production of NADH in TCA cycle, increased NADH utilization, and decrease in NADH/NAD+ ratio ? reduction of quinones ? reduction of O2 and production of O2-, H2O2, and HO* ? oxidative stress ? thiol stress ? metal stress ? membrane depolarization ? energy depletion ? inhibition of biosynthesis ? death. These events could be sequential or some could be parallel, which will be determined in this project. This project has three Specific Aims, which will test the predictions of each of the above events in this proposed model of PGRP killing, using Escherichia coli and Bacillus subtilis as model bacteria. Aim 1 will determine that each of these proposed events actually happens during PGRP killing of bacteria (some of these events are only predictions). Aim 2 will determine which of these events participate in PGRP-induced killing and which in bacterial defense against killing, or which are a consequence of killing. Aim 3 will determine the sequence of these events in PGRP-induced killing and which events are sequential and which parallel. The expected outcome of this project will be identification of the sequence of events responsible for PGRP-induced bacterial killing and for bacterial defense against killing, and the mechanism of their induction. The health-related significance of this research will be elucidation how human innate immunity proteins kill bacteria and a possibility of future development of new approaches to antimicrobial therapy, aimed at activating the killing systems and inhibiting the defense systems in bacteria. This approach could be effective against antibiotic-resistant bacteria, which is an urgently growing problem in the United States and worldwide.